Prothrombin and fibrinogen conversion during aggregation of αIIbβ3-inhibited platelets. Washed platelets from healthy volunteers pretreated with dRGDW for 5 minutes (200 μM) were activated with collagen (4 μg/mL), a mixture of factors VIIa (1.2 μg/mL), X (10 μg/mL), II (20 ng/mL), and fibrinogen (0.5 mg/mL), or with collagen in combination with the coagulation factors, all in the presence of 3 mM CaCl2. Incubations took place in aggregometry cuvettes at 37°C using a stir speed of 900 rpm. At different time points after initiation of αIIbβ3-independent aggregation, samples were taken and thrombin generation was quenched by addition of EDTA (50 mM) and hirudin (5 U/mL). After centrifugation, the prothrombin fragment 1 + 2 (A) or the fibrinogen activation peptide FPA (B) were measured in the supernatant by ELISA. F1 + 2 and FPA generation by collagen and coagulation factors VIIa/X/II/I was also measured in the absence of platelets. A representative of 3 independent experiments is shown.
